Glutamate transport modulatory compounds and methods

ABSTRACT

Described herein are methods of modulating excitatory amino acid transporter (EAAT) protein expression, methods of treating disease and disease symptoms, methods of identifying compounds that modulate EAAT protein expression, and compounds useful for modulating EAAT protein expression and treating disease and disease symptoms.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 60/450,227, filed Feb. 26, 2003. The entire contents of thisapplication is incorporated herein by this reference.

GOVERNMENT SUPPORT

This work described herein was supported by a grant from the NationalInstitutes of Health (Grant No. NS33958). Therefore, the U.S. Governmentmay have certain rights in the invention.

BACKGROUND OF THE INVENTION

Neurological disorders can significantly impact the central nervoussystem (CNS) and motor neuron units. For example, certain neurologicaldisorders of the CNS are known to adversely affect the brain andassociated structures. Neurological disorders affecting motor neuronunits have been grouped into motor neuron diseases and peripheralneuropathies. See generally Kandel, E. R. et al; (1991) in Principles ofNeuroscience, Appleton & Lange, Norwalk, Conn.; and Rowland, L. P. (ed.)(1982) in Human Motor Neuron Diseases. New York. Raven Press.

An illustrative motor neuron disease is amyotrophic lateral sclerosis(ALS). ALS has been reported to be a chronic neuromuscular disorderhaving recognized clinical manifestations. For example, it has beensuggested that degeneration of cortical and spinal/bulbar motor neuronsmay play a key role in the disorder. ALS is nearly always fatal. About95% of all ALS cases are sporadic, with many of the remaining casesshowing autosomal dominant inheritance. See e.g., Kuncl R. W. et al.,(1992) Motor Neuron Diseases In Diseases of the Nervous System, Asburyet al. eds. (Philadelphia W.B. Saunders) pp. 1179-1208; Brown, R. H.,(1996) Amer. Neurol. 30:145; Siddique, T. and Deng., H. X. (1996) Hum.Mol. Genetics 5:1465).

Specific CNS disorders have been also described. In particular, somehave been attributed to cholinergic, dopaminergic, adrenergic,serotonergic deficiencies or combinations thereof CNS disorders ofsevere impact include pre-senile dementia (sometimes referred to asAlzheimer's disease (AD) or early-onset Alzheimer's disease), seniledementia (dementia of the Alzheimer's type), Parkinson's disease (PD),and Huntington's disease (HD, sometimes referenced as Huntington'schorea). Such CNS disorders are well-represented in the humanpopulation. See generally; Gusella, J. F. et al. (1983) Nature 306: 234;Borlauer. W. and Jprmuloewoca. P. (eds.) (1976); Adv. in Parkinsonism:Biochemistry, Physiology, Treatment. Fifth International Symposium onParkinson's Disease (Vienna) Basel: Roche; and references cited therein.

Significant attention has been directed towards understanding theetiology of motor neuron diseases. For example, abnormal levels ofcertain excitotoxic neurotransmitters have been reported to adverselycontribute to many motor neuron diseases. In particular,glutamate-mediated excitotoxicity is recognized to have a critical rolein ALS. See e.g., Rothstein J. D. et al., (1990) Ann. Neurol. 28: 18;Rothstein J. D. et al. (1992) N. Engl. Med. 326: 1464; Rothstein J. D.et al. (1993) PNAS (USA) 90: 6591; and Lacomblez, L. et al., (1996)Lancet 347: 1179.

The astroglial transporters GLAST (EAAT1) and GLT-1 (EAAT2) areresponsible for the largest percentage of glutamate transport in theforebrain. As such, they both represent intriguing targets formodulation of expression, and thereby as agents to retard diseaseprogression, including neurodegeneration, seizure, and brain tumorgrowth.

There has been substantial efforts towards understanding mechanisms forreducing glutamate levels in the nervous system. For example,high-affinity, sodium-dependent glutamate transport is one reportedmeans of inactivating glutamate. In particular, astrocytic excitatoryamino acid transporter 2 (EAAT2) proteins are believed to havesubstantial functions in that inactivation. See e.g., Rothstein J. D. etal. (1994) Neuron 28: 18; Rothstein J. D. et al., (1995) Ann. Neurol.38: 78. and references cited therein.

In particular, investigations have suggested that EAAT2 is a predominantglutamate transporter. More particularly, certain antisense knockdownstudies have been reported to demonstrate that EAAT2 loss can lead toexcitotoxic neuronal degeneration and progressive motor impairment.Studies of ALS and other neurodegenerative disorders have relatedimpaired glutamate transport to loss of the EAAT2 protein. Inparticular, up to 60% to 70% of the sporadic ALS patients examined havea 30% to 95% loss of the EAAT2 protein. See e.g., Haugeto et al., supra;Rothstein J. D., et al., (1996) Neuron 16: 675; Bristol, L. A. andRothstein, J. D. (1996) Ann. Neurol. 39: 676.

There have been attempts to treat or prevent neurological disorders ofthe CNS and the motor neuron units. However, most existing therapies donot always stem the development or severity of the disorders inafflicted patients. See e.g., Rowell, (1987) Adv. Behav. Biol. 31: 191;Rinne, et al. Brain Res. (1991) 54: 167; U.S. Pat. No. 5,210,076 toBerliner; Yurek, D. M. (1990) Ann. Rev. Neurosci. 13: 415, and Rowlandet al. supra.

Accordingly, there is a need in the field for effective therapies fortreating neurological disorders.

SUMMARY OF THE INVENTION

Described herein are methods of modulating excitatory amino acidtransporter (EAAT) protein expression, methods of treating disease anddisease symptoms, methods of identifying compounds that modulate EAATprotein expression, and compounds useful for modulating EAAT proteinexpression and treating disease and disease symptoms.

In one aspect, the invention relates to a method of increasing EAAT2protein expression including the step of contacting a cell with at leastone EAAT2 expression promoting agent. In another aspect, the EAAT2expression promoting agent is a compound identified by a screening assayincluding the steps of

a) contacting the nucleic acid molecule comprising a cDNA molecule andnucleotide sequence which is at least about 60% identical to thenucleotide sequence of SEQ ID NO:1, 2, 3, or 4, wherein the nucleic acidmolecule is capable of directing mRNA expression from a promoterlessreporter vector, or a complement thereof, or a cell comprising saidnucleic acid molecule, with a test compound; and

b) determining whether expression of the mRNA or the polypeptide encodedby the cDNA is modulated, thereby identifying a compound which modulatesexpression of the mRNA or the polypeptide encoded by the cDNA as acompound which is capable of treating a neurological or psychiatricdisorder. The method can be wherein EAAT2 protein expression isincreased in vivo or wherein EAAT2 protein expression is increased invitro.

Other aspects of the methods are those wherein the EAAT2 expressionpromoting agent is an antibiotic, an anti-hypertensive, aneurotransmitter, an antibacterial, an anti-inflammatory, steroidderivative, or an anti-septic; those wherein the EAAT2 expressionpromoting agent comprises at least one structural element selected fromheterocycles having at least one ring sulfur atom, tertiary amines,quaternary ammonium salts, steroids, polyols, polyketide, guanidine,urea, or arsenate; those, wherein the EAAT2 expression promoting agentinclude at least one structural element selected from tertiary amines,quaternary ammonium salts, polyketides, steroidal ring systems andheterocycles having one or two rings, at least one sulfur ring atom or0, 1, or 2 nitrogen ring atoms; those wherein the EAAT2 expressionpromoting agent increases EAAT2 production by 200% or more relative tonon-regulated production; wherein the EAAT2 expression promoting agentincreases EAAT2 production by 300% or more relative to non-regulatedproduction; wherein the EAAT2 expression promoting agent increases EAAT2production by 600% or more relative to non-regulated production.

In another aspect, the methods are those delineated herein wherein theEAAT2 expression promoting agent is selected from the group consistingof penicillin V potassium, dequalinium chloride, quinapril, amoxicillin,pridinol methansulfonate, aklomide, vancomycin hydrochloride,thiaphenicol, ceftriaxone sodium, 1,3-dipropyl-8-cyclopentylxanthine(DPCPX), cephapirin sodium, actinospectacin, cefoperazole sodium,acivicin, acetylcholine, chloramphenicol, vidarabine, atenolol,oxytetracycline, glafenine, oxymetazoline hydrochloride, gallamine,perillic acid (−), amitriptyline hydrochloride, tetracainehydrochloride, disopyramide phosphate, sisomicin sulfate, ketaminehydrochloride, xylazine, bicuculline, flurbiprofen, cefadroxil,bacampicillin hydrochloride, tiapride hydrochloride, norethindroneacetate, bergaptene, carisoprodol, citiolone, piroxicam, erythromycinethylsuccinate, furegrelate sodium, albendazole, dihydrostreptomycinsulfate, aloin, fenoprofen, flutamide, ampicillin sodium, amprolium,sparteine sulfate, medroxyprogesterone acetate, alexidine hydrochloride,clindamycin hydrochloride, cephalothin sodium, daidzein, meclizinehydrochloride, lindane, bromopride,N-(3-trifluoromethylphenyl)piperazine hydrochloride (TFMPP), enoxolone,ipratropium bromide, bufexamac, gluconolactone, rifampin,hydroxychloroquine, coleoforsin, chloroxine, oxidopamine hydrochloride,camptothecin, nafcillin sodium, mianserin hydrochloride, acetarsol,prilocaine hydrochloride, deferoxamine mesylate, hexamethonium bromide,methenamine, paraxanthine, harmalol hydrochloride, pyrithione zinc,hydrocortisone butyrate, acetazolamide, aminoglutethimide, meclofenoxatehydrochloride, 2-phenpropylamino-5-nitrobenzoic acid (NPPB), amiodaronehydrochloride, aconitine, hydroxyprogesterone caproate, and diosmin.

In another aspect, the methods are those delineated herein, wherein theEAAT2 expression promoting agent is selected from the group consistingof penicillin V potassium, dequalinium chloride, quinapril, amoxicillin,pridinol methansulfonate, aklomide, vancomycin hydrochloride,thiaphenicol, ceftriaxone sodium, 1,3-dipropyl-8-cyclopentylxanthine(DPCPX), cephapirin sodium, actinospectacin, cefoperazole sodium,acivicin, acetylcholine, chloramphenicol, vidarabine, atenolol,oxytetracycline, glafenine, and oxymetazoline hydrochloride; thosewherein the EAAT2 expression promoting agent is selected from the groupconsisting of penicillin V potassium, dequalinium chloride, quinapril,amoxicillin, pridinol methansulfonate, aklomide, and vancomycinhydrochloride; those wherein the EAAT2 expression promoting agent isselected from the group consisting of penicillin V potassium,dequalinium chloride, and quinapril.

Another aspect is a method for decreasing extracellular glutamateconcentration in a mammal, the method including the step ofadministering at least one EAAT2 expression promoting agent to themammal. The expression promoting agent can be a compound identified by ascreening assay including the steps of:

a) contacting the nucleic acid molecule comprising a cDNA molecule andnucleotide sequence which is at least about 60% identical to thenucleotide sequence of SEQ ID NO:1, 2, 3, or 4, wherein the nucleic acidmolecule is capable of directing mRNA expression from a promoterlessreporter vector, or a complement thereof, or a cell comprising saidnucleic acid molecule, with a test compound; and

b) determining whether expression of the mRNA or the polypeptide encodedby the cDNA is modulated, thereby identifying a compound which modulatesexpression of the mRNA or the polypeptide encoded by the cDNA as acompound which is capable of treating a neurological or psychiatricdisorder. The method can be that wherein EAAT2 protein expression isincreased in vivo; or that wherein EAAT2 protein expression is increasedin vitro. In these methods, the EAAT2 expression promoting agent is anantibiotic, an anti-hypertensive, a neurotransmitter, and antibacterial,an anti-inflammatory, steroid derivative, or anti-septic; the EAAT2expression promoting agent includes at least one structural elementselected from heterocycles having at least one ring sulfur atom,tertiary amines, quaternary ammonium salts, steroids, polyols,polyketide, guanidine, urea, or arsenate; the EAAT2 expression promotingagent having at least one structural element selected from tertiaryamines, quaternary ammonium salts, polyketide, steroidal ring systemsand heterocycles having one or two rings, at least one sulfur ring atomand 0, 1, or 2 nitrogen ring atoms.

In another aspect, the methods herein are those wherein the mammal is aprimate; and those wherein the mammal is a human.

The methods delineated herein are also those wherein the extracellularglutamate concentration is reduced by at least about 50% relativenon-regulated concentration; or those wherein the extracellularglutamate concentration is reduced by at least about 75% relativenon-regulated concentration.

Another aspect is a method of treating a mammal suffering from orsusceptible to a disease or disorder associated with altered glutamatetransmission, the method including the step of administering to themammal a therapeutic amount of at least one EAAT expression promotingagent capable of increasing EAAT2 expression. The disease or disorderassociated with altered glutamate transmission can be a neurologicaldisease (e.g., Parkinson's disease, Huntington's disease, Alzheimer'sdisease, multiple sclerosis, amyotrophic lateral sclerosis, acuteneurological diseases, epilepsy, spinal cord injury, brain trauma,glaucoma, or psychiatric disorders. The method can be those wherein theEAAT2 expression promoting agent is a compound identified by a screeningassay comprising the steps of:

a) contacting the nucleic acid molecule comprising a cDNA molecule andnucleotide sequence which is at least about 60% identical to thenucleotide sequence of SEQ ID NO:1, 2, 3, or 4, wherein the nucleic acidmolecule is capable of directing mRNA expression from a promoterlessreporter vector, or a complement thereof, or a cell comprising saidnucleic acid molecule, with a test compound; and

b) determining whether expression of the mRNA or the polypeptide encodedby the cDNA is modulated,

thereby identifying a compound which modulates expression of the mRNA orthe polypeptide encoded by the cDNA as a compound which is capable oftreating a neurological or psychiatric disorder.

In another aspect, the invention relates to any of the methodsdelineated herein wherein the EAAT2 expression promoting agent is acompound identified by a screening assay including the steps of

a) contacting a cell that expresses EAAT2, with a test compound; and

b) determining whether expression of the EAAT2 in the cell is modulatedin the presence of the test compound compared to in the absence of thetest compound,

thereby identifying a compound which modulates expression of the EAAT2as a compound which is capable of treating a neurological or psychiatricdisorder. The method can be that wherein the EAAT2 expression promotingagent is a β-lactam antibiotic; or that wherein the EAAT2 expressionpromoting agent is a penicillin class, cephalosporin class, carbapenamclass or monobactam class compound.

Another aspect is a method of treating a mammal to modulate glutamateneurotransmission, the method including administering to the mammal atherapeutically effective amount of at least one EAAT expressionpromoting agent capable of increasing EAAT2 expression. In otheraspects, the methods are those wherein the mammal is in need oftreatment for a condition that is associated with learning or memory, orthose wherein the administration is for enhancing learning, memory; orcognitive enhancement.

The methods herein include administering to the subject (including asubject identified as in need of such treatment) an effective amount ofa compound described herein, or a composition described herein toproduce such effect. Identifying a subject in need of such treatment canbe in the judgment of a subject or a health care professional and can besubjective (e.g. opinion) or objective (e.g. measurable by a test ordiagnostic method).

Table 1 lists compounds (or salts or solvates thereof) useful in themethods delineated herein. TABLE 1 β-lactam compounds Cephalosporins andOther Beta Penicillins cephamycins Lactams benzylpenicillin (penicilling) cefaclor Aztreonam procaine benzylpenicillin cefadroxil Imipenem(procaine penicillin) cefadyl Meropenem phenoxymethylpenicillincefalexin Ertapenem (penicillin v) cefamandole FK-037 benzathinepenicillin cefazolin hetacillin cefditoren cloxacillin cefepimecarbenicillin cefetamet flucloxacillin cefdinir ampicillin cefiximeamoxicillin cefizox co-amoxiclav cefotaxime carboxypenicillincefmetazole ticarcillin cefobid timentin cefonicid tazocin(ureidopenicillin cefoperazone piperacillin with the beta- cefotanlactamase inhibitor tazobactam) cefotetan piperacillin cefoxitinpivmecillinam cefpirome amoxicillin-clavulanate cefpodoxime piperacillincefpodoxime proxetil oxacillin cefprozil cefradine ceftazidimeceftibuten ceftidoren ceftin ceftizoxime ceftriaxone cefuroximecefuroxime axetil cephalexin cefzil cephalothin

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is sequence listings for SEQ ID NOs: 1-4, which are EAAT2promoter sequences.

FIG. 2A shows spinal cord cultures incubated with test compound; 2B is asample slot blot from tissue homogenates; 2C illustrates arepresentative screening slot blot; 2D illustrates screening results ofa library of test compounds; 2E is an illustration of expression resultsfrom treatment with various compounds categorized by classes; 2F shows adose-response analysis of EAAT2 expression for ceftriaxone.

FIG. 3A-3E illustrate expression of EAAT2 promoter fragments in mousebrain; 3F shows astrocytes from EAAT1 promoter reporter, and 3G corticalexpression, in transgenic mice.

FIG. 4A shows activation (by compound class) of EAAT2 promoter byvarious test compounds; 4B illustrates dose-response results.

FIG. 5A is a western blot of ceftriaxone effect on GLT-1 and GLT-1Bexpression; 5B illustrates the effect of ceftriaxone on GLT-1 and GLT-1Bexpression; 5C is a western blot of ceftriaxone effect on GLAST, EAAC1and EAAT4 expression; 5D illustrates the effect of ceftriaxone on GLAST,EAAC1 and EAAT4 expression.

FIG. 6 illustrates the effect of various antibiotics on glutamatetransport.

FIG. 7A illustrates the effect of ceftriaxone on ischemic tolerance; 7Billustrates the effect of ceftriaxone on motor neuron degeneration; 7Cillustrates the effect of ceftriaxone on grip strength (in vivo model);7D illustrates the effect of ceftriaxone on survival in G93A mice.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, at least in part, on the discovery ofthe sequence of the EAAT2 promoter. Accordingly, the present inventionprovides nucleic acid molecules comprising the EAAT2 promoter, as wellas screening assays useful for identifying compounds which modulate theactivity of the EAAT2 promoter, and methods of treating neurological andpsychiatric disorders comprising administration of EAAT2 promotermodulators.

The acidic amino acids glutamate (Glu) and aspartate are the predominantexcitatory neurotransmitters in the mammalian central nervous system(CNS). Although there are millimolar concentrations of these excitatoryamino acids (EAAs) in the brain, extracellular concentrations aremaintained in the low micromolar range to facilitate crisp synaptictransmission and to limit the neurotoxic potential of these EAAs. Afamily of Na⁺-dependent high affinity transporters is responsible forthe regulation and clearance of extracellular EAAs.

Glutamate and aspartate activate ligand-gated ion channels that arenamed for the agonists N-methyl-D-aspartate (NMDA),a-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), and kainate.These ionotropic EAA receptors mediate rapid synaptic depolarization andare important for a number of other physiological processes, includingsynaptic plasticity and synapse development. The EAAs also activate afamily of metabotropic receptors coupled through G-proteins to secondmessenger systems or ion channels. It is well established that the EAAsare extremely important for normal brain function. However, there issubstantial evidence that an extracellular accumulation of EAAs andexcessive activation of EAA receptors also contributes to the neuronalcell death observed in acute insults to the CNS. The process known as,‘excitotoxicity’, may also contribute to neuronal loss observed inchronic neurodegenerative diseases, including amyotrophic lateralsclerosis (ALS).

The intracellular concentrations of glutamate (5-10 mM) and aspartate(1-5 mM) are 1000-fold to 10,000-fold greater than the extracellularconcentrations (<1-10 μM). Unlike many other neurotransmitters, there isno evidence that glutamate or aspartate is metabolized extracellularly.Instead, they are cleared from the extracellular space by transport intoneurons and astrocytes.

Several subtypes of Na⁺-dependent glutamate transporters have beenidentified through pharmacological strategies and cDNA cloning. Fiveknown distinct cDNA clones that express Na⁺-dependent high-affinityglutamate transport are referred to herein as GLT-1/EAAT2, EAAC1/EAAT3,GLAST/EAAT1, EAAT4, and EAAT5. There is also evidence for additionalheterogeneity of GLT-1 and GLAST that originates from alternate mRNAsplicing.

Expression of two of these transporters, GLT-1 and GLAST, is generallyrestricted to astroglia. Expression of two other transporters, EAAC1 andEAAT4, is generally restricted to neurons, and EAAT5 is thought to berestricted to retina. Of the three transporters found in forebrain(GLT-1, GLAST, and EAAC1), GLT-1 appears to be the only transporter thatis specific to brain tissue, suggesting that GLT-1 expression iscontrolled by brain specific mechanisms.

Previously, it was thought that presynaptic transporters had a majorrole in the clearance of EAAs during synaptic transmission. This wasbased on the evidence that activity is enriched 2-fold in synaptosomalmembrane preparations compared to fractions enriched in mitochondria ormyelin. However, it is now known that these membrane preparationscontain resealed glial membranes and tremendous amounts of GLT-1protein. In addition, it has long been known that lesions of specificafferents result in a decrease in Na⁺-dependent transport in targetareas. For example, lesions of the cortical projections to the striatumresult in decreased uptake in striatal synaptosomes. These types ofstudies suggested that there was significant transport into presynapticterminals, but more recent studies have suggested that these lesionsreduce expression of the glial transporters.

Evidence from several complementary strategies strongly suggests thatGLT-1 mediates the bulk of Na⁺-dependent transport of EAAs in the CNS.For example, the pharmacological properties of GLT-1 parallel thepredominant component of activity observed in rat brain membranes. Basedon the enrichment required to purify GLT-1 to homogeneity, it is thoughtthat GLT-1 represents approximately 1% of total brain protein. Selectiveimmunoprecipitation of GLT-1 from solubilized forebrain tissue andreconstitution of the remaining protein in liposomes, suggests thatGLT-1 mediates 90% of transport activity. Anti-sense knock-down of GLT-1results in the dramatic reductions in synaptosomal transporter activityin several forebrain regions. Synaptosomal uptake in mice geneticallydeleted of GLT-1 is 5% of normal. Finally, electrophysiologicalrecording of transporter mediated currents in brain preparationsstrongly suggest that GLT-1 has a primary role for the clearance ofglutamate during synaptic transmission in several forebrain regions.

The expression of GLT-1/EAAT2 is dynamically regulated both in vivo andin vitro. Although GLT-1 is the predominant transporter in the adultCNS, expression is rather low early in development and increases duringsynaptogenesis in both rats and humans. As described above, lesions ofprojections to a particular target nucleus results in decreasedexpression of both glial transporters, GLT-1 and GLAST. These datasuggest that the presence of neurons induces and/or maintains expressionof the glial transporters.

Several different groups have demonstrated decreased expression of GLT-1and/or GLAST in animal models of acute insults to the CNS, includingstroke and traumatic brain injury. A loss in GLT-1 expression has beendemonstrated in patients with ALS. Furthermore, there is evidence ofdecreased expression of these transporters in humans with chronicneurodegenerative diseases, including Alzheimer's Disease, andHuntington's Disease. Loss of GLT-1 is also a feature of the fatal braintumor, glioblastoma multiforma.

Amyotrophic lateral sclerosis (ALS) is the most common form of adultmotor neuron disease in which there is progressive degeneration of boththe upper motor neurons in the cortex and the lower motor neurons in thebrain stem and spinal cord. The majority of ALS cases (95%) areapparently sporadic (SALS), while approximately 5% are familial (FALS).Cleveland D W, Rothstein J D, Nat. Rev. Neurosci. (2001); 2:806-819;Kuncl R W, Crawford T O, Rothstein J D, Drachman D B. Motor neurondiseases. In: Asbury A K et al., editors. Diseases of the NervousSystem. 2 ed. Philadelphia: W.B. Saunders, 1992:1179-1208. FALS caseswere found to be associated with mutations in SOD-1. Andersen P M et al.Genetics of amyotrophic lateral sclerosis: an overview. In: Brown R H,Jr., Meininger V, Swash M, editors. Amyotrophic lateral sclerosis.London: Martin Dunitz, 2000:223-250; Brown R H, Jr. Amyotrophic lateralsclerosis. Insights from genetics. Arch Neurol 1997; 54:1246-1250;Cleveland D W, Rothstein J D. Nat. Rev. Neurosci. 2001; 2:806-819, thegene that encodes copper-zinc superoxide dismutase (CuZnSOD). SOD1mutations account for about 15-20% of all FALS. SOD1 mutations have beenused to generate transgenic mouse models; G93A, G37R, G86R and G85R SOD1all produce reliable motor neuron degeneration in transgenic miceover-expressing the mutant protein. Cleveland D W., Neuron 1999;24:515-520; Cleveland D W et al., Nature 1995; 378:342-343; Cleveland DW, Rothstein J D. Nat. Rev. Neurosci. 2001; 2:806-819. Pathogenic events“downstream” of mutant SOD1 toxicity include excitotoxicity,neuroinflammation and apoptosis. Increasingly, these downstream eventshave been the target of pharmacotherapy—in some cases successfullyaltering disease course. Multiple other genes or chromosomallocalization have been identified in other familial variants of ALS.

Common to both familial and sporadic ALS is the loss of the astroglialglutamate transporter EAAT2 protein. As described above, the astroglialtransporter EAAT2 is the predominant protein responsible for the bulk ofsynaptic clearance of glutamate. In particular, EAAT2 protects againstexcitotoxic neurodegeneration. Evidence of abnormalities in glutamatehandling initially arose in ALS from discovery of large increases incerebral fluid levels of glutamate in ALS patients, findings nowreported in ˜40% of sporadic ALS patients. Rothstein J D et al. Ann.Neurol. 1990; 28:18-25; Spreux-Varoquaux O et al. J Neurol Sci. 2002;193:73-78. Measurement of functional glutamate transport in ALS tissuerevealed a marked diminution in the affected ALS brain regions. The lossof functional glutamate transporter is likely the result of a dramaticloss of astroglial glutamate transporter protein EAAT2, which can be inup to 65% of sporadic ALS patients. Rothstein J D et al., Ann. Neurol.1994; 36:282; Rothstein J D, et al. N Engl. J Med 1992; 326:1464-1468;Rothstein J D et al., Ann Neurol 1995; 38:73-84. Regardless of themechanism, lowering EAAT2 with antisense oligonucleotides hasdemonstrated that loss of transport activity directly provokes neuronaldeath. Furthermore, expression of at least three (G85R, G93A, G37R)SOD1mutants in transgenic mice—all lead to a loss of the EAAT2 protein andits function. Rothstein J D et al., Neuron 1996; 16:675-686; Rothstein JD et al., Proc Natl Acad Sci USA 1993; 90:6591-6595. In aggregate, theseand other studies studies suggest that that the functional loss of EAAT2(associated with astrocyte dysfunction), contributes to the loss ofmotor neurons in both inherited and sporadic ALS. Recently, we alsodocumented a loss of the GLT-1/EAAT2 protein in a new rat transgenicmodel of the disease. Howland D S et al., Proc. Natl. Acad. Sci. U.S.A2002; 99:1604-1609. Notably, loss of transporter protein precedes actualdegeneration of motor neurons and their axons in the rat model.

Two labs recently provided important data as to the importance of EAAT2as a therapeutic. Dr. Glen Lin, reported that a 2 fold overexpression ofEAAT2, in transgenic mice, leads to neuroprotection in vitro, anddelayed onset of disease in ALS mice. Guo, H. et al. Hum Mol Genet.2003; 12:2519-2532. Similarly, Dr. Margaret Sutherland, has reportedthat a five fold over expression of EAAT2 in transgenic mice, canincrease survival of G93A SOD1 mice by at least 30 days (and in severalanimals many months longer). Maguire J L, et al. Soc Neurosci Abstr.2001; 27:607.9; Sutherland M. L., et al. Soc Neurosci Abs. 2003. Inaddition, her lab has reported that increased EAAT2 can also attenuateseizures and significantly diminished both seizures and tumor growth inglioma xenografted rodents. As will be shown below, we have alsogenerated data that suggest that over expression of EAAT2 can delaydisease onset in ALS mice, using novel therapeutics.

Even though GLT-1 expression is extremely high in vivo, ‘normal’astrocytes maintained in culture express essentially no detectable mRNAor protein. Co-culturing astrocytes with neurons induces glialexpression of GLT-1, suggesting that neurons induce and/or maintainexpression of GLT-1 in vitro. This effect of neurons is, at least inpart, mediated by a soluble secreted molecule. Several small moleculesmimic this effect of neurons, including dbcAMP, epidermal growth factor,pituitary adenylate cyclase-activating peptide, and immunophilin. In allof these cases the increases in GLT-1 protein expression are accompaniedby an increase in GLT-1 mRNA and a change in the morphology of theastrocytes that many believe are reminiscent of differentiation.

The effects of dbcAMP are blocked by an inhibitor of protein kinase A.It has been shown that the increase in GLT-1 expression induced bydbcAMP, epidermal growth factor, or neuron conditioned medium are allblocked by an inhibitor of either phosphatidylinositol 3-kinase or aninhibitor of the transcription factor NF-κB. Otherwise, little is knownabout the mechanisms that actually control GLT-1 expression. Thus, theidentification of the EAAT2 promoter provides a valuable tool tounderstand EAAT2 regulation and to develop assays to control itssynthesis.

As used herein, the term “EAAT2” refers to the human astroglialglutamate transporter 2 gene. See, e.g., U.S. Pat. No. 5,658,782 whichdiscloses the human EAAT2 cDNA sequence, the disclosure of the which isspecifically incorporated herein by reference. As used herein, the term“GLT-1” refers to the rodent astroglial glutamate transporter 2 gene.

As used herein, the term “promoter” generally refers a region of genomicDNA, usually found 5′ to an mRNA transcription start site. Promoters areinvolved in regulating the timing and level of mRNA transcription andcontain, for example, binding sites for cellular proteins such as RNApolymerase and other transcription factors. As used interchangeablyherein, the terms “EAAT2 promoter”, “EAAT2 promoter region” and the likeinclude the region of genomic DNA found 5′ to the EAAT2 mRNAtranscription start site. In preferred embodiments, the EAAT2 promotercomprises SEQ ID NO:1, 2, 3, or 4, or fragments thereof. When insertedinto a promoterless reporter construct, preferred EAAT2 promoterfragments are able to direct transcription of the reporter gene.

In one embodiment, the EAAT2 promoter includes SEQ ID NO:1 (e.g.,nucleotides 1-4696 of SEQ ID NO:1). In another embodiment the EAAT2promoter includes a P1 region, which comprises nucleotides 733-3450 ofSEQ ID NO:1 (also set forth as SEQ ID NO:2). In another embodiment, theEAAT2 promoter includes a P2 region, which comprises nucleotides733-3186 of SEQ ID NO:1 (also set forth as SEQ ID NO:3). In stillanother embodiment, the EAAT2 promoter includes a P3 region, whichcomprises nucleotides 2590-3450 of SEQ ID NO:1 (also set forth as SEQ IDNO:4).

The EAAT2 promoter activation molecules of the present invention providetherapeutic agents for neurological and psychiatric disorders. As usedherein, the term ‘neurological disorder’ includes a disorder, disease orcondition which affects the nervous system, e.g., the central nervoussystem. The neurological disorders that can be treated in accord withthe present invention include specific disorders that have been reportedto be associated with excitotoxicity. Particularly included arespecified neurological disorders affecting motor neuron function.Neurological disorders include, but are not limited to, amyotrophiclateral sclerosis (ALS), trinucleotide repeat expansion disorders (e.g.,Huntington's disease (HD), spinal and bulbar muscular atrophy,spinocerebellar ataxia types 1, 2, 6, and 7, dentatorubropallidoluysianatrophy, and Machado-Joseph disease), α-synucleinopathies (e.g.,Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiplesystem atrophy (MSA)), multiple sclerosis (MS), Alzheimer's disease,brain tumors (e.g., glioblastoma), stroke/ischemia, cerebrovasculardisease, epilepsy (e.g., temporal lobe epilepsy), HIV-associateddementia, Korsakoff's disease, chronic pain, neurogenic pain, painfulneuropathies, headaches (e.g., migraine headaches), Pick's disease,progressive supranuclear palsy, Creutzfeldt-Jakob disease, Bell's Palsy,aphasia, sleep disorders, glaucoma, and Meniere's disease.

In addition, the EAAT2 promoter activation molecules of the presentinvention provide therapeutic agents for modulation of normal glutamateneurotransmission associated with brain functions such as learning andmemory. The molecules described herein can be administered to a subjectin need of such treatment for the enhancement of memory and learning.

As used herein, the term ‘psychiatric disorder’ refers diseases anddisorders of the mind, and includes diseases and disorders listed in theDiagnostic and Statistical Manual of Mental Disorders—Fourth Edition(DSM-IV), published by the American Psychiatric Association, WashingtonD.C. (1994). Psychiatric disorders include, but are not limited to,anxiety disorders (e.g., acute stress disorder agoraphobia, generalizedanxiety disorder, obsessive-compulsive disorder, panic disorder,posttraumatic stress disorder, separation anxiety disorder, socialphobia, and specific phobia), childhood disorders, (e.g.,attention-deficit/hyperactivity disorder, conduct disorder, andoppositional defiant disorder), eating disorders (e.g., anorexia nervosaand bulimia nervosa), mood disorders (e.g., depression, bipolardisorder, cyclothymic disorder, dysthymic disorder, and major depressivedisorder), personality disorders (e.g., antisocial personality disorder,avoidant personality disorder, borderline personality disorder,dependent personality disorder, histrionic personality disorder,narcissistic personality disorder, obsessive-compulsive personalitydisorder, paranoid personality disorder, schizoid personality disorder,and schizotypal personality disorder), psychotic disorders (e.g., briefpsychotic disorder, delusional disorder, schizoaffective disorder,schizophreniform disorder, schizophrenia, and shared psychoticdisorder), substance-related disorders (e.g., alcohol dependence,amphetamine dependence, cannabis dependence, cocaine dependence,hallucinogen dependence, inhalant dependence, nicotine dependence,opioid dependence, phencyclidine dependence, and sedative dependence),adjustment disorder, autism, delirium, dementia, multi-infarct dementia,learning and memory disorders (e.g., amnesia and age-related memoryloss), and Tourette's disorder.

As noted, neurological and psychiatric disorders of specific interestinclude those associated with abnormal release or removal of excitotoxicamino acids such as glutamate. Several CNS neuron types are especiallyadversely affected by excitotoxic glutamate. See e.g., Choi, D. W.(1988) Neuron 1: 623; and references cited therein. Specificallypreferred neurological disorders include AD, HD, PD with ALS beingespecially preferred.

III. Screening Assays

The invention provides a method (also referred to herein as a “screeningassay”) for identifying modulators, i.e., candidate or test compounds oragents (e.g., nucleic acids, peptides, peptidomimetics, small molecules,or other drugs) which bind to the EAAT2 promoter, and/or which have astimulatory or inhibitory effect on, for example, EAAT2 promoteractivity.

In one embodiment, the invention provides assays for screening candidateor test compounds which are modulators EAAT2 promoter activity. Inanother embodiment, the invention provides assays for screeningcandidate or test compounds which bind to or modulate the activity of anEAAT2 promoter. The test compounds of the present invention can beobtained using any of the numerous approaches in combinatorial librarymethods known in the art, including: biological libraries; spatiallyaddressable parallel solid phase or solution phase libraries; syntheticlibrary methods requiring deconvolution; the ‘one-bead one-compound’library method; and synthetic library methods using affinitychromatography selection. The biological library approach is limited topeptide libraries, while the other four approaches are applicable topeptide, non-peptide oligomer or small molecule libraries of compounds(Lam, K. S. (1997) Anticancer Drug Des. 12:45).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example, in: DeWitt et al. (1993) Proc. Natl.Acad. USA 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al.(1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed.Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061;and Gallop et al. (1994) J. Med. Chem. 37:1233.

Libraries of compounds may be presented in solution (e.g., Houghten(992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (LadnerU.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. '409), plasmids(Cull et al. (1992) Proc. Natl. Acad. Sci. USA 89:1865-1869) or on phage(Scott and Smith (1990) Science 249:386-390); (Devlin (1990) Science249:404-406); (Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA87:6378-6382); (Felici (1991) J. Mol. Biol. 222:301-310); (Ladnersupra.).

In a preferred embodiment, an assay is a cell-based assay in which acell which expresses a reporter gene operatively linked to an EAAT2promoter or portion thereof (e.g., whose expression is under the controlof the EAAT2 promoter or portion thereof) is contacted with a testcompound and the ability of the test compound to modulate EAAT2 promoteractivity is determined. Determining the ability of the test compound tomodulate EAAT2 promoter activity can be accomplished by monitoringreporter gene expression (e.g., reporter mRNA or polypeptide expressionlevel) or activity, for example. As described elsewhere herein, thereporter can be any detectable marker. For example, the reporter can bea nucleic acid sequence, the expression of which can be measured by, forexample, Northern blotting, RT-PCR, primer extension, or nucleaseprotection assays. The reporter may also be a nucleic acid sequence thatencodes a polypeptide, the expression of which can be measured by, forexample, Western blotting, ELISA, or RIA assays. Reporter expression canalso be monitored by measuring the activity of the polypeptide encodedby the reporter using, for example, a standard glutamate transportassay, a luciferase assay, a β-galactosidase assay, a chloramphenicolacetyl transferase (CAT) assay, or a fluorescent protein assay.

The level of expression or activity of a reporter under the control ofthe EAAT2 promoter in the presence of the candidate compound is comparedto the level of expression or activity of the reporter in the absence ofthe candidate compound. The candidate compound can then be identified asa modulator of EAAT2 promoter activity based on this comparison. Forexample, when expression of reporter mRNA or protein expression oractivity is greater (statistically significantly greater) in thepresence of the candidate compound than in its absence, the candidatecompound is identified as a stimulator of EAAT2 promoter activity.Alternatively, when expression or activity of reporter mRNA or proteinis less (statistically significantly less) in the presence of thecandidate compound than in its absence, the candidate compound isidentified as an inhibitor of EAAT2 promoter activity.

The ability of the test compound to bind to the EAAT2 promoter and/or tomodulate the binding of proteins (e.g., transcription factors) to theEAAT2 promoter can also be determined. Determining the ability of thetest compound to bind to and/or modulate EAAT2 promoter binding to abinding protein can be accomplished, for example, by coupling the testcompound, the EAAT2 promoter or the binding protein with a radioisotopeor enzymatic label such that binding of the EAAT2 promoter to the testcompound or the binding protein can be determined by detecting thelabeled component in a complex. For example, compounds (e.g., the testcompound, the EAAT2 promoter, or a binding protein) can be labeled with³²P, ¹²⁵I, ³⁵S, ¹⁴C, or ³H, either directly or indirectly, and theradioisotope detected by direct counting of radioemission or byscintillation counting. Alternatively, compounds can be enzymaticallylabeled with, for example, horseradish peroxidase, alkaline phosphatase,or luciferase, and the enzymatic label detected by determination ofconversion of an appropriate substrate to product.

It is also within the scope of this invention to determine the abilityof a compound (e.g., a test compound or EAAT2 promoter binding protein)to interact with the EAAT2 promoter without the labeling of any of theinteractants. For example, a microphysiometer can be used to detect theinteraction of a compound with the EAAT2 promoter without the labelingof either the compound or the EAAT2 promoter (McConnell, H. M. et al.(1992) Science 257:1906-1912). As used herein, a “microphysiometer”(e.g., Cytosensor) is an analytical instrument that measures the rate atwhich a cell acidifies its environment using a light-addressablepotentiometric sensor (LAPS). Changes in this acidification rate can beused as an indicator of the interaction between a compound and the EAAT2promoter.

In another embodiment, the assay is a cell-free assay in which an EAAT2promoter or portion thereof is contacted with a test compound and theability of the test compound to modulate (e.g., stimulate or inhibit)the activity of the EAAT2 promoter or portion thereof is determined.Determining the ability of the test compound to modulate the activity ofan EAAT2 promoter can be accomplished, for example, by determining theability of the EAAT2 promoter to bind to an EAAT2 promoter targetmolecule by one of the methods described above for determining directbinding. Determining the ability of the EAAT2 promoter to bind to anEAAT2 promoter target molecule can also be accomplished using atechnology such as real-time Biomolecular Interaction Analysis (BIA).Sjolander, S. and Urbaniczky, C. (1991) Anal. Chem. 63:2338-2345 andSzabo et al. (1995) Curr. Opin. Struct. Biol. 5:699-705. As used herein,“BIA” is a technology for studying biospecific interactions in realtime, without labeling any of the interactants (e.g., BIAcore). Changesin the optical phenomenon of surface plasmon resonance (SPR) can be usedas an indication of real-time reactions between biological molecules.

In yet another embodiment, the cell-free assay involves contacting anEAAT2 promoter or portion thereof with a known compound which binds theEAAT2 promoter (e.g., a component of the basal transcription machinery)to form an assay mixture, contacting the assay mixture with a testcompound, and determining the ability of the test compound to interactwith the EAAT2 promoter, wherein determining the ability of the testcompound to interact with the EAAT2 promoter comprises determining theability of the EAAT2 promoter to preferentially bind to or modulate theactivity of an EAAT2 promoter target molecule.

In more than one embodiment of the above assay methods of the presentinvention, it may be desirable to immobilize either EAAT2 promoter orits target molecule to facilitate separation of complexed fromuncomplexed forms of one or both of the molecules, as well as toaccommodate automation of the assay. Binding of a test compound to anEAAT2 promoter, or interaction of an EAAT2 promoter with a substrate ortarget molecule in the presence and absence of a candidate compound, canbe accomplished in any vessel suitable for containing the reactants.Examples of such vessels include microtiter plates, test tubes, andmicro-centrifuge tubes. In one embodiment, a fusion protein can beprovided which adds a domain that allows one or both of the proteins tobe bound to a matrix. For example, glutathione-S-transferase/targetfusion proteins can be adsorbed onto glutathione sepharose beads (SigmaChemical, St. Louis, Mo.) or glutathione derivatized micrometer plates,which are then combined with the test compound or the test compound andeither the non-adsorbed target protein or EAAT2 promoter, and themixture incubated under conditions conducive to complex formation (e.g.,at physiological conditions for salt and pH). Following incubation, thebeads or microtiter plate wells are washed to remove any unboundcomponents, the matrix immobilized in the case of beads, complexdetermined either directly or indirectly, for example, as describedabove. Alternatively, the complexes can be dissociated from the matrix,and the level of EAAT2 promoter binding or activity determined usingstandard techniques.

Other techniques for immobilizing proteins or nucleic acids on matricescan also be used in the screening assays of the invention. For example,either an EAAT2 promoter or an EAAT2 promoter substrate or targetmolecule can be immobilized utilizing conjugation of biotin andstreptavidin. Biotinylated EAAT2 promoter, substrates, or targetmolecules can be prepared from biotin-NHS (N-hydroxysuccinimide) usingtechniques known in the art (e.g., biotinylation kit, Pierce Chemicals,Rockford, Ill.), and immobilized in the wells of streptavidin-coated 96well plates (Pierce Chemical). Alternatively, antibodies reactive withEAAT2 promoter or target molecules but which do not interfere withbinding of the EAAT2 promoter to its target molecule can be derivatizedto the wells of the plate, and unbound target or EAAT2 promoter trappedin the wells by antibody conjugation. Methods for detecting suchcomplexes, in addition to those described above for the GST-immobilizedcomplexes, include immunodetection of complexes using antibodiesreactive with the EAAT2 promoter or target molecule, as well asenzyme-linked assays which rely on detecting an enzymatic activityassociated with the EAAT2 promoter or target molecule.

In yet another aspect of the invention, the EAAT2 promoter can be usedas “bait” in a one-hybrid assay (see, e.g., BD Matchmaker One-HybridSystem (1995) Clontechniques X(3):2-4; BD Matchmaker LibraryConstruction & Screening Kit (2000) Clontechniques XV (4):5-7; BD SMARTtechnology overview (2002) Clontechniques XVII (1):22-28; Ausubel, F.M., et al. (1998 et seq.) Current Protocols in Molecular Biology Eds.Ausubel, F. M., et al., pp. 13.4.1-13.4.10) to identify proteins whichbind to or interact with the EAAT2 promoter (“EAAT2 promoter-bindingproteins” or “EAAT2 promoter-bp”) and are involved in EAAT2 promoteractivity. Such EAAT2 promoter-binding proteins are also likely to beinvolved in the regulation of transcription from the EAAT2 promoter.

In another aspect, the invention pertains to a combination of two ormore of the assays described herein. For example, a modulating agent canbe identified using a cell-based or a cell-free assay, and the abilityof the agent to modulate the activity of an EAAT2 promoter can beconfirmed in vivo, e.g., in an animal such as an animal model for aneurological disease.

This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein inan appropriate animal model (e.g., an animal model for a neurologicaldisease). For example, an agent identified as described herein (e.g., anEAAT2 promoter modulating agent or an EAAT2 promoter binding protein)can be used in an animal model to determine the efficacy, toxicity, orside effects of treatment with such an agent. Alternatively, an agentidentified as described herein can be used in an animal model todetermine the mechanism of action of such an agent. Furthermore, thisinvention pertains to uses of novel agents identified by theabove-described screening assays for treatments as described herein.

Agents are described herein that are identified using the screeningmethods delineated herein. These compounds include a variety of chemicalstructures as identified herein, including compounds having a β-lactamring system, more specifically, β-lactam antibiotic compounds, includingpenicillin class, cephalosporin class, carbapenam class and monobactamclass compounds.

IV. Methods of Treatment

In one embodiment, the present invention provides methods of treatingneurological and psychiatric disorders which comprise administering atherapeutically effective amount of a pharmaceutical compositioncomprising an EAAT2 promoter modulator a subject (e.g., a mammal such asa human).

To modulate EAAT2 promoter activity, and thereby modulate EAAT2 geneexpression, e.g., a compound disclosed herein or identified by thescreening assays of the invention, can be administered to a cell or asubject. Administration of an EAAT2 promoter modulator to mammaliancells (including human cells) can modulate (e.g., up- or down-regulateEAAT2 mRNA and/or polypeptide expression, thereby up- or down-regulatingglutamate transport into the cell. In such methods, the EAAT2 promotercan be administered to a mammal (including a human) by known procedures.

The preferred therapeutic methods of the invention (which includeprophylactic treatment) in general comprise administration of atherapeutically effective amount of an EAAT2 promoter modulator to ananimal in need thereof, including a mammal, particularly a human. Suchtreatment will be suitably administered to subjects, particularlyhumans, suffering from, having, susceptible to, or at risk for aneurological or psychiatric disorder. The EAAT2 promoter modulators ofthe invention may be also used in the treatment of any other disordersin which EAAT2 may be implicated.

For therapeutic applications, EAAT2 modulators of the invention may besuitably administered to a subject such as a mammal, particularly ahuman, alone or as part of a pharmaceutical composition, comprising theEAAT2 modulator together with one or more acceptable carriers thereofand optionally other therapeutic ingredients. The carrier(s) must be“acceptable” in the sense of being compatible with the other ingredientsof the formulation and not deleterious to the recipient thereof.

The pharmaceutical compositions of the invention include those suitablefor oral, rectal, nasal, topical (including buccal and sublingual),vaginal or parenteral (including subcutaneous, intramuscular,intravenous and intradermal) administration. The formulations mayconveniently be presented in unit dosage form, e.g., tablets andsustained release capsules, and in liposomes, and may be prepared by anymethods well know in the art of pharmacy. See, for example, Remington'sPharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa.(17th ed. 1985).

Such preparative methods include the step of bringing into associationwith the molecule to be administered ingredients such as the carrierwhich constitutes one or more accessory ingredients. In general, thecompositions are prepared by uniformly and intimately bringing intoassociation the active ingredients with liquid carriers, liposomes orfinely divided solid carriers or both, and then if necessary shaping theproduct.

Compositions of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, sachets or tabletseach containing a predetermined amount of the active ingredient; as apowder or granules; as a solution or a suspension in an aqueous liquidor a non-aqueous liquid; or as an oil-in-water liquid emulsion or awater-in-oil liquid emulsion, or packed in liposomes and as a bolus,etc.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active ingredient in afree-flowing form such as a powder or granules, optionally mixed with abinder, lubricant, inert diluent, preservative, surface-active ordispersing agent. Molded tablets may be made by molding in a suitablemachine a mixture of the powdered compound moistened with an inertliquid diluent. The tablets optionally may be coated or scored and maybe formulated so as to provide slow or controlled release of the activeingredient therein.

Compositions suitable for topical administration include lozengescomprising the ingredients in a flavored basis, usually sucrose andacacia or tragacanth; and pastilles comprising the active ingredient inan inert basis such as gelatin and glycerin, or sucrose and acacia.

Compositions suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents. The formulations may be presented in unit-dose or multi-dosecontainers, for example, sealed ampules and vials, and may be stored ina freeze dried (lyophilized) condition requiring only the addition ofthe sterile liquid carrier, for example water for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and tablets.

Application of the subject therapeutics often will be local, so as to beadministered at the site of interest. Various techniques can be used forproviding the subject compositions at the site of interest, such asinjection, use of catheters, trocars, projectiles, pluronic gel, stents,sustained drug release polymers or other device which provides forinternal access. Where an organ or tissue is accessible because ofremoval from the patient, such organ or tissue may be bathed in a mediumcontaining the subject compositions, the subject compositions may bepainted onto the organ, or may be applied in any convenient way.

It will be appreciated that actual preferred amounts of a given EAAT2modulator of the invention used in a given therapy will vary to theparticular active compound being utilized, the particular compositionsformulated, the mode of application, the particular site ofadministration, the patient's weight, general health, sex, etc., theparticular indication being treated, etc. and other such factors thatare recognized by those skilled in the art including the attendantphysician or veterinarian. Optimal administration rates for a givenprotocol of administration can be readily determined by those skilled inthe art using conventional dosage determination tests.

The invention will be further described in the following examples. Itshould be understood that these examples are for illustrative purposesonly and are not to be construed as limiting this invention in anymanner.

EXAMPLES Example 1 In Vitro Analysis of EAAT2 Protein Expression

Screening Assay for EAAT2 Protein overexpression. Spinal cordorganotypic cultures and astroglial cultures are used to screen fordrugs capable of stimulating EAAT2 synthesis and function. Organotypiccultures offer the advantage in that they maintain the normalarchitecture of neuron-astroglial interactions in vitro and are derivedfrom post natal tissue; thus may better reflect astroglial responses invivo (rather than embryonic cells). Thus a drug that acts either on anastrocyte—or induces neurons to secrete factors that alertastrocytes—better reflects the “natural” condition of delivering a drugto a whole animal.

Bioassay Method (see FIG. 2 for assay description summary)

Organotypic Spinal Cord. Spinal cord organotypic cultures have beendescribed by us in detail in the past. Rothstein J D et al., N. Engl. J.Med. 1992; 326:1464-1468. Briefly, 300 um sections of rat lumbar spinalcord, from postnatal day 8-10 rat pups, are placed on MilliporeMillicell CM semipermeable membranes. Each well contains 5 slices (FIG.2A). Fifty-100 cultures can be prepared weekly. Each drug (10-100 μM)was added for 3 days, along with cell culture medium/serum. Cultureswere harvested and 5-50 μg of tissue was applied to slot blot apparatusfor detection of EAAT2 by standard Western blotting/chemiluminescencemethods described in the past. Kuncl R W et al., Motor neuron diseases.In: Asbury A K et al., editors. Diseases of the Nervous System. 2 ed.Philadelphia: W.B. Saunders, 1992:1179-1208; Rothstein J D et al.,Neuron 1994; 13:713-725. All antipeptide antibodies were affinitypurified and highly specific for transporter subtypes. A typicalslot-blot analysis, is shown in FIG. 2B,C. By this method, we canreliably detect increases greater than 50% of expressed protein. Foreach antibody slot blot, the homogenates used are expected to be withinthe linear range for antibody detection, based on prior standard curves.

Screening Library-Assay Design. The library of compounds for these firststudies was the NINDS Custom Collection from Microsource Discovery. Thelibrary is composed of 1040 compounds in 96 well plates, that alsoincluded positive control for transporter synthesis (dibutyryl cyclicAMP [dbcAMP], GDNF). The library is a unique collection of knownbioactive compounds that permit the simultaneous evaluation of hundredsof marketed drugs and biochemical standards. Each compound was studiedat a final concentration of 10-100 μM. All assays were performed induplicate. A typical slot blot is shown in FIG. 2C.

Data Analysis. All blots were analyzed by laser densitometry (BioRadImage Quant) and the duplicate points were averaged. The complete resultdataset from the 1040 compounds is shown in FIG. 2D. Each blot includeda positive control standard (e.g. dbcAMP) and a negative controlstandard (e.g. serum, DMSO). Data was kept in Excel Spreadsheets, usinga numerical/text coding system. All positive drugs (positive defined asat least a 50% increased in protein expression) were reevaluated.

RESULTS: Screened Drugs Can Increase EAAT2 In vitro. After screening1040 compounds, we were able to identify more than 10 related compoundscapable of increasing EAAT2 protein levels by 3.5 to 7 fold (see FIG.2E). In total, we identified 80 compounds capable of increasing EAAT2 by2 fold or more in the first screen. Of that list, β-lactam antibioticswere overly represented and were the most common structural motifobserved in all compounds—15 different beta lactam antibiotics wereactive. As shown in FIG. 2E, these β-lactams were all capable ofincreased EAAT2 protein expression. A follow-up dose response analysis(FIG. 2F) revealed and EC₅₀ for protein expression for ceftriaxone of3.5 μM.

Example 2 EAAT2 Promoter Reporter Activation

Generation of COS7 and Human Astroglial Promoter Reporter Cell Lines.

EAAT2 promoter (E2P) isolation and Reporter Generation. A 2.7 kb EAAT2promoter fragment was obtained by cutting the PAC clone RP4-683L5 withKpn1 and Nco1. Previous studies document that sequence, 5′ of EAAt2coding region, has promoter motifs and can be activated in vitro. Thepromoter was cloned into the pGL3-basic luciferase reporter vector(Promega) (refered to as pE2P-GL3) or pEGFP-1 plasmid (Clontech) (namedpE2P-eGFP). E2P was also cloned into a pLck-eGFP plasmid (amyristoylated version of eGFP that targets the eGFP protein to themembrane). Finally, E2P was also cloned into apE2P-Luciferase-IRES-Lck-eGFP plasmid, which has the fragmentE2P-Luciferase from pE2P-GL3, followed by an IRES (internal ribosomalentry site), followed by Lck-eGFP (named pELILE). In this lastconstruct, E2P drives the expression of both Luciferase and eGFP at thesame time.

Generation of E2P-eGFP and Bac-EAAT1-eGFP Transgenic mice (FIG. 3). Toprovide screening cell lines for the assays we have now successfullygenerated two transgenic mice that express the EAAT2 promoter fragment(E2P) or the full length EAAT1 promoter (Bac-EAAT1). As shown in FIG. 3we have generated E2P transgenic mice that demonstrate widespreadexpression of the EAAT2 promoter reporter in the CNS. Similar wegenerated Bac-EAAt1 mice and expressing cells. Recently the Heintz groupalso generated an EAAT1 Bac-reporter based mouse based on a similar Bacconstruct used in our own mice.

Screening assays. Lipofectamine 2000 reagent was used to transfect Cos-7and HEK-293 cells. Human cortical astroglial cell were obtained from ourcollegue, Dr. Avi Nath. The EpE2P-eGFP, pE2P-Lck-eGFP, and pELILEcontain a Neomycin resistance gene that permitted establishment ofstable cell lines. For the human astroglial cells, SV40 was used toimmortalize the cell. Stably transfected cells were seeded on 24-wellplates, incubated with 10 uM compound solution for 48 hours and thefluorescence intensity was recorded with an automated reader(SpectraGeminiXS).

RESULTS: Identification of EAAT2 Promoter activating compounds (FIG. 4).From the original NINDS screen, we identified numerous β lactamcompounds capable of potently activating EAAt2 promoter. As shown inFIG. 4, most β-lactams were able to increase EAAT promoter—far more thanthe known positive control, dibutyrl cyclic AMP. All compounds wereactive at a pharmacologically relevant concentration of 1-10 uM—aconcentration range that these compounds can be found in the CNS afterstandard anti-bacterial therapy (e.g. ceftriaxone).

Example 3 In Vivo Activation of EAAT Expression/Function

In vivo activation of protein expression/function can be assessed asdelineated in the example below using ceftriaxone as the test compound.

Ceftriaxone Increases Brain GLT1 level (FIG. 5). To determine if a drugidentified in Phases 1 and 2 could actually induce EAAT expression invivo, we administered ceftriaxone to rats (n=5) (and mice, n=3) daily.Ceftriaxone was administered at a dose known to lead to CNS levels, 200mg/kg ip. After 5 days of chronic daily administration animals weresacrificed and brain tissue harvested. As shown in FIG. 5A,B,ceftriaxone therapy lead to 3 fold increase in brain GLT1 levels, aswell as its normal splice product, GLT1b. This increase is comparable tothe promoter activation results seen in vitro (FIG. 4). Western blotsfor the astroglial glutamate transporter GLAST as well as the twoneuronal glutamate transporters, EAAC1 and EAAT4, showed no alterationin transporter expression after ceftriaxone therapy (FIG. 5C,D).Similarly, the constitutive protein, actin, was unchanged by ceftriaxoneadministration (FIG. 5A,C).

Ceftriaxone Increases Brain GLT1 level (FIG. 5). To determine if a drugidentified in Phases 1 and 2 could actually induce EAAT expression invivo, we administered ceftriaxone to rats (n=5) (and mice, n=3) daily.Ceftriaxone was administered at a dose known to lead to CNS levels, 200mg/kg ip. After 5 days of chronic daily administration animals weresacrificed and brain tissue harvested. As shown in FIG. 5A,B,ceftriaxone therapy lead to 3 fold increase in brain GLT1 levels, aswell as its normal splice product, GLT1b. This increase is comparable tothe promoter activation results seen in vitro (FIG. 4). Was this effecttransporter specific? Western blots for the astroglial glutamatetransporter GLAST as well as the two neuronal glutamate transporters,EAAC1 and EAAT4, showed no alteration in transporter expression afterceftriaxone therapy (FIG. 5C,D). Similarly, the constitutive protein,actin, was unchanged by ceftriaxone administration (FIG. 5A,C).

Example 4 Neuroprotection of Compounds

To evaluate the potential neuroprotection afforded by increasedexpression of EAAT2 by promoter activating drugs, we have conductedseveral in vitro and in vivo experiments—where glutamate toxicitycontributes to neuronal death. Neuroprotection can be assessed asdelineated in the example below using β-lactam antibiotics as the testcompound.

In Vitro Model of Ischemia—Oxygen glucose deprivation (FIG. 7A) The invitro model of oxygen glucose deprivation (OGD) is a well known and wellaccepted model of acute neural injury. In our in-vitro model ofischemia, one hour of oxygen glucose deprivation (OGD) is lethal tocultured neurons, with toxicity known to involve excess glutamate.However, when these cultures are preconditioned 24 hours prior to thelethal condition with transient OGD (5 minutes), there is a dramatic androbust resistance of neurons to cell death. The data indicate that thisneuroprotection may be due, in part, to increased expression of GLT1.

Method. Primary cortical mixed neuronal-glial cell cultures are preparedfrom gestation day 14-16 CD1 mice. The preparation of these culturesfrom mouse fetal cortex is well-described. Experiments are performed atdays in vitro 13-15. In the experimental condition, cultures aresubjected to oxygen glucose deprivation (OGD), an in-vitro model ofischemia. Cortical cells are either subjected to control treatment(media, modified Earle's balanced salt solution including glucose andbubbled with 5% CO₂ 95% O₂, is changed alongside treatment groups, butno OGD is performed), or 5 minutes (sublethal) of OGD (using modifiedEarle's balanced salt solution which is devoid of glucose and bubbledwith 10% H₂, 85% N₂, and 5% to deoxygenate). Anaerobic conditions areachieved using an anaerobic chamber at 37° C. OGD is terminated byexchange of media back to oxygenated growing medium. Twenty-four hoursfollowing the above, cortical cells are subjected either to notreatment, or one hour of OGD. Neuronal survival is determined bycomputer-assisted cell counting after staining with the fluorescentvital dyes propidium iodide (as an indicator of neuronal death) andHoechst 33342 (as an indicator of total number of neurons) and ispresented as percent of cell death. Glial nuclei fluoresce at a lowerintensity and are gated out. Drugs are added (Ceftriaxone 1 μM) 24 hoursprior to the first experimental condition, and thus have been in theculture medium 48 hours prior to onset of 1 hour OGD. Following 1 hourOGD, cells are returned to growing medium without drugs.

Ceftriaxone Neuroprotection. Baseline neuronal death in the cultures is14%, as shown in the no treatment column (NT) of FIG. 7A. Data arepresented as average neuronal death in separate wells of one experiment.1 μM Ceftriaxone, when added for 48 hours in these cultures, does notincrease the baseline cell death (NT+Ceftriaxone). When cultures aresubjected to 1 hour OGD, neuronal cell death, as expected, increasesdramatically to approximately 50%. When cultures are preconditioned with5 minutes of OGD 24 hours prior to 1 hour OGD, percent cell death iscomparable to no treatment condition, indicating ischemic tolerance ofneurons in this condition. This is the well known phenomenon of ischemictolerance. Importantly, 1 μM Ceftriaxone, when added 48 hours prior to 1hour OGD, also protects neurons from cell death, reducing the percentageof neuronal cell death from 50% to 20%. (similar to ischemic toleranceneuroprotection). Thus, ceftriaxone pretreatment appears to preventneuronal death in ischemic tolerance.

In vitro model of chronic motor neurodegeneration (FIG. 7B). A model ofchronic neurodegeneration was used, based on the blockade of glutamatetransport in spinal cord organotypic cultures, with the non specificinhibitor threo-hytdroyxaspartate (THA) or TBOA. Chronic incubation ofcultures with THA (or TBOA) leads to chronic increase in extra cellularglutamate and subsequent slow death of motor neurons (over 4 weeks). Theorganotypic spinal cord culture model was developed to study aspects ofglutamate-mediated toxicity (and therapy). It has been useful inpre-clinical drug identification (including—riluzole—the only FDAapproved drug for ALS, and more recently—celecoxib). Increasedexpression of glutamate transporter GLT1, by genetic over expression(e.g. transfection or transgenic over expression), in this system, canprevent motor neuron death (not shown) and neuronal death in transgenicanimals. Guo H., et al. Hum Mol Genet. 2003; 12:2519-2532.

To determine if drug induced GLT1 promoter activation, and thesubsequent over expression of GLT1 protein could be neuroprotective, weused the organotypic spinal cord paradigm. Organotypic spinal cordcultures were prepared from lumbar spinal cords of 8-day-old rat pups,as described previously. Rothstein J D, et al. Proc Natl Acad Sci USA.1993; 90:6591-6595. Ceftriaxone was added with media changes. No drugswere added for the first 7 days following culture preparation. THA wasthen added to experimental cultures at a concentration of 100 μM, whichproduces death of motor neurons within 3 to 4 weeks. Variousconcentrations of ceftriaxone were added as indicated, to achieve finalconcentrations from 0 to 100 μM. Experiments were always performed withcontrol spinal cord cultures (ie—no drugs added), THA alone, ceftriaxonealone, and ceftriaxone+THA. Experiments at each concentration ofceftriaxone were repeated 3-5 times. The medium, with THA andceftriaxone at the indicated concentrations, was changed twice a week.After 4 weeks, cultures were fixed, and immuostained for neurofilament(SMI-32, Sternberger) to quantify large ventral horn motor neurons (awell established method to follow motor neuron survival in this system).

Neuroprotection by ceftriaxone. As shown in FIG. 7B, ceftriaxonetreatment prevented motor neurons loss in a dose dependent manner. ASshown in Preliminary Data—Phases 1 and 2, this concentration ofceftriaxone increases GLT1 protein and function by at least 3 fold.Importantly, the concentrations used in these studies are within therange attainable with oral/parenteral administration of ceftriaxone (1-4grams/day). Notably, neuroprotection cannot be seen in cultures preparedfrom GLT-1 null mice (not shown).

In Vivo Neuroprotection—Effect of Ceftriaxone on Onset and Progressionof Motor Neuron Disease in the G93A SOD1 Mouse. (FIG. 7C,D)

To determine if ceftriaxone could alter neurodegeneration in a diseasemodel that involves altered expression of glutamate transporters wetreated G93A SOD1 mice with ceftriaxone. Numerous studies havedocumented a contributory role for excess glutamate in this mouse model-and role for modulating glutamate receptors or transporters inneuroprotective strategies. Guo H., et al. Hum Mol Genet. 2003;12:2519-2532. Modest over expression—by a transgenic approach—can alterdisease onset and/or survival. Furthermore, recent studies suggest thatlate administration of drugs, e.g. at time of disease onset, may be moretherapeutically relevant.

Treatment paradigm. G93A SOD1 mice [(B6.Cg-Tg(SOD1-G93A)1Gur/J, highexpresser] were treated with ceftriaxone (200 mg/kg ip) starting atapproximately 12 weeks of age. Drug treated animals (n=20) and salineinjected controls (n=20) were monitored daily for survival and weeklyfor grip strength (Columbus Instruments) and for body weight, asdescribed previously.

Ceftriaxone delays loss of Grip Strength and Increases survival. Asshown in FIG. 7C, ceftriaxone treatment significantly delayed loss ofmuscle strength. This effect was observed within 7 days after treatment,and persisted for 4 weeks, By 18 weeks of age the strength preservationwas lost. In a similar manner, the drug also increased over all survivalof the mice by about 7-10 days (FIG. 7D). Although this effect isrelatively small, the drug was given at the time of disease onset, andthus, even a small effect may have clinical significance. Theneuroprotection seen in this study is not likely to be due to the normalantibiotic properties of the drug—since mice have no known infections at12-16 weeks of age—when prominent muscle strength effects were seen.

All references cited herein, whether in print, electronic, computerreadable storage media or other form, are expressly incorporated byreference in their entirety, including but not limited to, abstracts,articles, journals, publications, texts, treatises, technical datasheets, internet web sites, databases, patents, patent applications, andpatent publications.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

1. A method of increasing EAAT2 protein expression comprising the stepof contacting a cell with at least one EAAT2 expression promoting agent.2. The method of claim 1, wherein the EAAT2 expression promoting agentis a compound identified by a screening assay comprising the steps of:a) contacting a test compound with a nucleic acid molecule comprising acDNA molecule and having a nucleotide sequence which is at least about60% identical to the nucleotide sequence of SEQ ID NO:1, 2, 3, or 4,wherein the nucleic acid molecule is capable of directing mRNAexpression from a promoterless reporter vector, or a complement thereof,or a cell comprising said nucleic acid molecule; and b) determiningwhether expression of the mRNA or the polypeptide encoded by the cDNA ismodulated, thereby identifying a compound which is an EAAT2 expressionpromoting agent.
 3. The method of either claim 1 wherein EAAT2 proteinexpression is increased in vivo.
 4. The method of either claim 1 whereinEAAT2 protein expression is increased in vitro.
 5. The method of claim1, wherein the EAAT2 expression promoting agent is an antibiotic, ananti-hypertensive, a neurotransmitter, an antibacterial, ananti-inflammatory, a steroid derivative, or an anti-septic.
 6. Themethod of claim 1, wherein the EAAT2 expression promoting agentcomprises at least one structural element selected from heterocyclescomprising at least one ring sulfur atom, tertiary amines, quaternaryammonium salts, steroids, polyols, polyketide, guanidine, urea, orarsenate.
 7. The method of claim 6, wherein the EAAT2 expressionpromoting agent comprises at least one structural element selected fromtertiary amines, quaternary ammonium salts, polyketides, steroidal ringsystems and heterocycles having one or two rings, at least one sulfurring atom and 0, 1, or 2 nitrogen ring atoms.
 8. The method of claim 1,wherein the EAAT2 expression promoting agent increases EAAT2 productionby 200% or more relative to non-regulated EAAT2 production.
 9. Themethod of claim 8, wherein the EAAT2 expression promoting agent isselected from the group consisting of penicillin V, penicillin Vpotassium, dequalinium chloride, quinapril, amoxicillin, pridinolmethansulfonate, aklomide, vancomycin hydrochloride, thiaphenicol,ceftriaxone, ceftriaxone sodium, 1,3-dipropyl-8-cyclopentylxanthine,cephapirin sodium, actinospectacin, cefoperazole, cefoperazole sodium,acivicin, acetylcholine, chloramphenicol, vidarabine, atenolol,oxytetracycline, glafenine, oxymetazoline hydrochloride, gallamine,perillic acid (−), amitriptyline hydrochloride, tetracainehydrochloride, disopyramide phosphate, sisomicin sulfate, ketaminehydrochloride, xylazine, bicuculline, flurbiprofen, cefadroxil,bacampicillin hydrochloride, tiapride hydrochloride, norethindroneacetate, bergaptene, carisoprodol, citiolone, piroxicam, erythromycinethylsuccinate, furegrelate sodium, albendazole, dihydrostreptomycinsulfate, aloin, fenoprofen, flutamide, ampicillin, ampicillin sodium,amprolium, sparteine sulfate, medroxyprogesterone acetate, alexidinehydrochloride, clindamycin hydrochloride, cephalothin, cephalothinsodium, daidzein, meclizine hydrochloride, lindane, bromopride,N-(3-trifluoromethylphenyl)piperazine hydrochloride, enoxolone,ipratropium bromide, bufexamac, gluconolactone, rifampin,hydroxychloroquine, coleoforsin, chloroxine, oxidopamine hydrochloride,camptothecin, nafcillin sodium, mianserin hydrochloride, acetarsol,prilocaine hydrochloride, deferoxamine mesylate, hexamethonium bromide,methenamine, paraxanthine, harmalol hydrochloride, pyrithione zinc,hydrocortisone butyrate, acetazolamide, aminoglutethimide, meclofenoxatehydrochloride, 2-phenpropylamino-5-nitrobenzoic acid, amiodaronehydrochloride, aconitine, hydroxyprogesterone caproate, and diosmin. 10.The method of claim 1, wherein the EAAT2 expression promoting agentincreases EAAT2 production by 300% or more relative to non-regulatedEAAT2 production
 11. The method of claim 10, wherein the EAAT2expression promoting agent is selected from the group consisting ofpenicillin V, penicillin V potassium, dequalinium chloride, quinapril,amoxicillin, pridinol methansulfonate, aklomide, vancomycinhydrochloride, thiaphenicol, ceftriaxone, ceftriaxone sodium,1,3-dipropyl-8-cyclopentylxanthine, cephapirin sodium, actinospectacin,cefoperazole sodium, acivicin, acetylcholine, chloramphenicol,vidarabine, atenolol, oxytetracycline, glafenine, and oxymetazolinehydrochloride.
 12. The method of claim 1, wherein the EAAT2 expressionpromoting agent increases EAAT2 production by 400% or more relative tonon-regulated EAAT2 production.
 13. The method of claim 12, wherein theEAAT2 expression promoting agent is selected from the group consistingof penicillin V, penicillin V potassium, dequalinium chloride,quinapril, amoxicillin, pridinol methansulfonate, aklomide, andvancomycin hydrochloride.
 14. The method of claim 1, wherein the EAAT2expression promoting agent increases EAAT2 production by 600% or morerelative to non-regulated EAAT2 production.
 15. The method of claim 14,wherein the EAAT2 expression promoting agent is selected from the groupconsisting of penicillin V, penicillin V potassium, dequaliniumchloride, and quinapril.
 16. A method for decreasing extracellularglutamate concentration in a mammal, the method comprising the step ofadministering at least one EAAT2 expression promoting agent to themammal.
 17. The method of claim 16, wherein the mammal has beenidentified as in need of such treatment.
 18. The method of claim 16,wherein the EAAT2 expression promoting agent is a compound identified bya screening assay comprising the steps of: a) contacting a test compoundwith a nucleic acid molecule comprising a cDNA molecule and having anucleotide sequence which is at least about 60% identical to thenucleotide sequence of SEQ ID NO:1, 2, 3, or 4, wherein the nucleic acidmolecule is capable of directing mRNA expression from a promoterlessreporter vector, or a complement thereof, or a cell comprising saidnucleic acid molecule; and b) determining whether expression of the mRNAor the polypeptide encoded by the cDNA is modulated, thereby identifyinga compound which is an EAAT2 expression promoting agent.
 19. The methodof claim 16, wherein EAAT2 protein expression is increased in vivo. 20.The method of claim 16, wherein EAAT2 protein expression is increased invitro.
 21. The method of claim 16, wherein the EAAT2 expressionpromoting agent is an antibiotic, an anti-hypertensive, aneurotransmitter, an antibacterial, an anti-inflammatory, a steroidderivative, or an anti-septic.
 22. The method of claim 16, wherein theEAAT2 expression promoting agent comprises at least one structuralelement selected from heterocycles comprising at least one ring sulfuratom, tertiary amines, quaternary ammonium salts, steroids, polyols,polyketide, guanidine, urea, or arsenate.
 23. The method of claim 22,wherein the EAAT2 expression promoting agent comprises at least onestructural element selected from tertiary amines, quaternary ammoniumsalts, polyketide, steroidal ring systems and heterocycles having one ortwo rings, at least one sulfur ring atom and 0, 1, or 2 nitrogen ringatoms.
 24. The method of claim 16, wherein the EAAT2 expressionpromoting agent increases EAAT2 production by 200% or more relative tonon-regulated EAAT2 production.
 25. The method of claim 24, wherein theEAAT2 expression promoting agent is selected from the group consistingof penicillin V, penicillin V potassium, dequalinium chloride,quinapril, amoxicillin, pridinol methansulfonate, aklomide, vancomycinhydrochloride, thiaphenicol, ceftriaxone, ceftriaxone sodium,1,3-dipropyl-8-cyclopentylxanthine, cephapirin sodium, actinospectacin,cefoperazole, cefoperazole sodium, acivicin, acetylcholine,chloramphenicol, vidarabine, atenolol, oxytetracycline, glafenine,oxymetazoline hydrochloride, gallamine, perillic acid (−), amitriptylinehydrochloride, tetracaine hydrochloride, disopyramide phosphate,sisomicin sulfate, ketamine hydrochloride, xylazine, bicuculline,flurbiprofen, cefadroxil, bacampicillin hydrochloride, tiapridehydrochloride, norethindrone acetate, bergaptene, carisoprodol,citiolone, piroxicam, erythromycin ethylsuccinate, furegrelate sodium,albendazole, dihydrostreptomycin sulfate, aloin, fenoprofen, flutamide,ampicillin, ampicillin sodium, amprolium, sparteine sulfate,medroxyprogesterone acetate, alexidine hydrochloride, clindamycinhydrochloride, cephalothin, cephalothin sodium, daidzein, meclizinehydrochloride, lindane, bromopride,N-(3-trifluoromethylphenyl)piperazine hydrochloride, enoxolone,ipratropium bromide, bufexamac, gluconolactone, rifampin,hydroxychloroquine, coleoforsin, chloroxine, oxidopamine hydrochloride,camptothecin, nafcillin sodium, mianserin hydrochloride, acetarsol,prilocaine hydrochloride, deferoxamine mesylate, hexamethonium bromide,methenamine, paraxanthine, harmalol hydrochloride, pyrithione zinc,hydrocortisone butyrate, acetazolamide, aminoglutethimide, meclofenoxatehydrochloride, 2-phenpropylamino-5-nitrobenzoic acid, amiodaronehydrochloride, aconitine, hydroxyprogesterone caproate, and diosmin. 26.The method of claim 16, wherein the EAAT2 expression promoting agentincreases EAAT2 production by 300% or more relative to non-regulatedEAAT2 production.
 27. The method of claim 25, wherein the EAAT2expression promoting agent is selected from the group consisting ofpenicillin V, penicillin V potassium, dequalinium chloride, quinapril,amoxicillin, pridinol methansulfonate, aklomide, vancomycinhydrochloride, thiaphenicol, ceftriaxone, ceftriaxone sodium,1,3-dipropyl-8-cyclopentylxanthine, cephapirin sodium, actinospectacin,cefoperazole sodium, acivicin, acetylcholine, chloramphenicol,vidarabine, atenolol, oxytetracycline, glafenine, and oxymetazolinehydrochloride.
 28. The method of claim 16, wherein the EAAT2 expressionpromoting agent increases EAAT2 production by 400% or more relative tonon-regulated EAAT2 production.
 29. The method of claim 28, wherein theEAAT2 expression promoting agent is selected from the group consistingof penicillin V, penicillin V potassium, dequalinium chloride,quinapril, amoxicillin, pridinol methansulfonate, aklomide, andvancomycin hydrochloride.
 30. The method of claim 16, wherein the EAAT2expression promoting agent increases EAAT2 production by 600% or morerelative to non-regulated EAAT2 production.
 31. The method of claim 30,wherein the EAAT2 expression promoting agent is selected from the groupconsisting of penicillin V, penicillin V potassium, dequaliniumchloride, and quinapril.
 32. The method of claim 16, wherein the mammalis a primate.
 33. The method of claim 32, wherein the mammal is a human.34. The method of claim 16, wherein the extracellular glutamateconcentration is reduced by at least about 50% relative to non-regulatedextracellular glutamate concentration.
 35. The method of claim 16,wherein the extracellular glutamate concentration is reduced by at leastabout 75% relative non-regulated concentration.
 36. A method of treatinga mammal suffering from or susceptible to a disease or disorderassociated with altered glutamate transmission, the method comprisingthe step of administering to the mammal a therapeutic amount of at leastone EAAT2 expression promoting agent capable of increasing EAAT2expression.
 37. The method of claim 36, wherein the mammal has beenidentified as in need of such treatment.
 38. The method of claim 36,wherein the disease or disorder associated with altered glutamatetransmission is a neurological disease.
 39. The method of claim 38,wherein the neurological disease is selected from the group consistingof Parkinson's disease, Huntington's disease, Alzheimer's disease,multiple sclerosis, amyotrophic lateral sclerosis, acute neurologicaldiseases, epilepsy, spinal cord injury, brain trauma, glaucoma, andpsychiatric disorders.
 40. The method of claim 36, wherein the EAAT2expression promoting agent is a compound identified by a screening assaycomprising the steps of a) contacting a test compound with a nucleicacid molecule comprising a cDNA molecule and having a nucleotidesequence which is at least about 60% identical to the nucleotidesequence of SEQ ID NO:1, 2, 3, or 4, wherein the nucleic acid moleculeis capable of directing mRNA expression from a promoterless reportervector, or a complement thereof, or a cell comprising said nucleic acidmolecule; and b) determining whether mRNA expression or the polypeptideencoded by the cDNA is modulated, thereby identifying a compound whichis an EAAT2 expression promoting agent.
 41. The method of claim 36,wherein the EAAT2 expression promoting agent is an antibiotic, ananti-hypertensive, a neurotransmitter, an antibacterial, ananti-inflammatory, a steroid derivative, or an anti-septic.
 42. Themethod of claim 36, wherein the EAAT2 expression promoting agentcomprises at least one structural element selected from heterocyclescomprising at least one ring sulfur atom, tertiary amines, quaternaryammonium salts, steroids, polyols, polyketides, guanidine, urea, orarsenate.
 43. The method of claim 42, wherein the EAAT2 expressionpromoting agent comprises at least one structural element selected fromtertiary amines, quaternary ammonium salts, polyketides, steroidal ringsystems and heterocycles having one or two rings, at least one sulfurring atom and 0, 1, or 2 nitrogen ring atoms.
 44. The method of claim 36wherein the EAAT2 expression promoting agent increases EAAT2 productionby 200% or more relative to non-regulated EAAT2 production.
 45. Themethod of claim 44, wherein the EAAT2 expression promoting agent isselected from the group consisting of penicillin V, penicillin Vpotassium, dequalinium chloride, quinapril, amoxicillin, pridinolmethansulfonate, aklomide, vancomycin hydrochloride, thiaphenicol,ceftriaxone, ceftriaxone sodium, 1,3-dipropyl-8-cyclopentylxanthine,cephapirin sodium, actinospectacin, cefoperazole, cefoperazole sodium,acivicin, acetylcholine, chloramphenicol, vidarabine, atenolol,oxytetracycline, glafenine, oxymetazoline hydrochloride, gallamine,perillic acid (−), amitriptyline hydrochloride, tetracainehydrochloride, disopyramide phosphate, sisomicin sulfate, ketaminehydrochloride, xylazine, bicuculline, flurbiprofen, cefadroxil,bacampicillin hydrochloride, tiapride hydrochloride, norethindroneacetate, bergaptene, carisoprodol, citiolone, piroxicam, erythromycinethylsuccinate, furegrelate sodium, albendazole, dihydrostreptomycinsulfate, aloin, fenoprofen, flutamide, ampicillin, ampicillin sodium,amprolium, sparteine sulfate, medroxyprogesterone acetate, alexidinehydrochloride, clindamycin hydrochloride, cephalothin, cephalothinsodium, daidzein, meclizine hydrochloride, lindane, bromopride,N-(3-trifluoromethylphenyl)piperazine hydrochloride, enoxolone,ipratropium bromide, bufexamac, gluconolactone, rifampin,hydroxychloroquine, coleoforsin, chloroxine, oxidopamine hydrochloride,camptothecin, nafcillin sodium, mianserin hydrochloride, acetarsol,prilocaine hydrochloride, deferoxamine mesylate, hexamethonium bromide,methenamine, paraxanthine, harmalol hydrochloride, pyrithione zinc,hydrocortisone butyrate, acetazolamide, aminoglutethimide, meclofenoxatehydrochloride, 2-phenpropylamino-5-nitrobenzoic acid, amiodaronehydrochloride, aconitine, hydroxyprogesterone caproate, and diosmin. 46.The method of claim 36, wherein the EAAT2 expression promoting agentincreases EAAT2 production by 300% or more relative to non-regulatedproduction.
 47. The method of claim 46, wherein the EAAT2 expressionpromoting agent is selected from the group consisting of penicillin V,penicillin V potassium, dequalinium chloride, quinapril, amoxicillin,pridinol methansulfonate, aklomide, vancomycin hydrochloride,thiaphenicol, ceftriaxone, 1,3-dipropyl-8-cyclopentylxanthine,cephapirin sodium, actinospectacin, cefoperazole, cefoperazole sodium,acivicin, acetylcholine, chloramphenicol, vidarabine, atenolol,oxytetracycline, glafenine, and oxymetazoline hydrochloride.
 48. Themethod of claim 36, wherein the EAAT2 expression promoting agentincreases EAAT2 production by 400% or more relative to non-regulatedEAAT2 production.
 49. The method of claim 48, wherein the EAAT2expression promoting agent is selected from the group consisting ofpenicillin V, penicillin V potassium, dequalinium chloride, quinapril,amoxicillin, pridinol methansulfonate, aklomide, and vancomycinhydrochloride.
 50. The method of claim 36, wherein the EAAT2 expressionpromoting agent increases EAAT2 production by 600% or more relative tonon-regulated EAAT2 production.
 51. The method of claim 50, wherein theEAAT2 expression promoting agent is selected from the group consistingof penicillin V, penicillin V potassium, dequalinium chloride, andquinapril.
 52. The method of claim 36, wherein the mammal is a primate.53. The method of claim 52, wherein the mammal is a human.
 54. Themethod of claim 36, wherein the extracellular glutamate concentration isreduced by at least about 50% relative non-regulated concentration. 55.The method of claim 35, wherein the extracellular glutamateconcentration is reduced by at least about 75% relative non-regulatedconcentration.
 56. The method of claim 36, wherein the EAAT2 expressionpromoting agent is a compound identified by a screening assay comprisingthe steps of: a) contacting a cell that expresses EAAT2, with a testcompound; and b) determining whether expression of the EAAT2 in the cellis modulated in the presence of the test compound compared to in theabsence of the test compound, thereby identifying a compound whichmodulates expression of the EAAT2.
 57. The method of claim 36, whereinthe EAAT2 expression promoting agent is a β-lactam antibiotic.
 58. Themethod of claim 36, wherein the EAAT2 expression promoting agent is apenicillin class, cephalosporin class, carbapenam class or monobactamclass compound.
 59. A method of treating a mammal to modulate glutamateneurotransmission, the method comprising administering to the mammal atherapeutically effective amount of at least one EAAT2 expressionpromoting agent capable of increasing EAAT2 expression.
 60. The methodof claim 59, wherein the mammal has been identified as in need of suchtreatment.
 61. The method of claim 59, wherein the mammal is in need oftreatment for a condition that is associated with learning or memory.62. The method of claim 61, wherein the administration is for enhancinglearning, memory; or cognition.
 63. The method of claim 16, wherein theEAAT2 expression promoting agent is a compound identified by a screeningassay comprising the steps of a) contacting a cell that expresses EAAT2with a test compound; and b) determining whether expression of the EAAT2in the cell is modulated in the presence of the test compound comparedto in the absence of the test compound, thereby identifying a compoundwhich modulates expression of the EAAT2.
 64. The method of claim 16,wherein the EAAT2 expression promoting agent is a β-lactam antibiotic.65. The method of claim 16, wherein the EAAT2 expression promoting agentis a penicillin class, cephalosporin class, carbapenam class ormonobactam class compound.
 66. The method of claim 36, wherein the EAAT2expression promoting agent is a compound identified by a screening assaycomprising the steps of a) contacting a cell that expresses EAAT2 with atest compound; and b) determining whether expression of the EAAT2 in thecell is modulated in the presence of the test compound compared to inthe absence of the test compound, thereby identifying a compound whichmodulates expression of the EAAT2.
 67. The method of claim 36, whereinthe EAAT2 expression promoting agent is a β-lactam antibiotic.
 68. Themethod of claim 36, wherein the EAAT2 expression promoting agent is apenicillin class, cephalosporin class, carbapenam class or monobactamclass compound.